Human epidermal growth factor receptor 2 (HER2), is overexpressed on ~30%of breast cancers and is the target of an FDA approved immunotherapeutic (trastuzumab). Since many antibody therapies, including trastuzumab, are most effective when used in conjunction with chemotherapy, direct attachment of a toxin to an antibody should increase their tumoricidal properties, while reducing the side-effects associated with system-wide toxin distribution. In this proposal an unnatural amino acid (UAA)incorporated in a Herceptin fragment (Fab)will be cross-linked to a known apoptosis initiator, tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), and the adduct screened against known trastuzumab-sensitive target cells. The use of a UAA in this system permits strict control over the number and placement of TRAIL binding groups, by allowing coupling through an orthogonally reactive linking molecule (a bi-functional polyethylene glycol, PEG),while permitting cross-linking to any hydroxylamine-functional molecule. A single UAA labeled antibody can therefore be coupled to any number of effector molecules to create combinatorial libraries of binder-effector complexes, allowing rapid development of antibody therapies. The linker methodology can be further extended to create multimeric structures of Fabs without the need for extensive re-engineering. These bivalent and multimeric Fab constructs will be tested agains cancer cell lines, and further tested for activity in tumor mouse models. Specific Aim 1: Engineer a TRAIL-anti-HER2 bivalent molecule 1A. Express UAA containing anti-HER2 and couple to TRAIL through orthogonal chemistry 1B. Test efficacy of cell killing on cell lines and in rodent models. Specific Aim 2: Generate anti-HER2 Fab multimers 2A. Express UAA containing (anti-HER2)2 and self dimerize 2B. Test effect of multimerization on tumor cell killing in vitro and in vivo in rodent models.. Since antibodies have been shown to effectively target some cancers without binding to normal cells, antibodies can be used to deliver toxins directly to tumor cells without destroying healthy cells. This proposal develops the tools to cross link antibodies to toxins using unique chemistries that can be further extended to other more toxin/antibody combinations in a rapid and simpleway.